Abstract

Most nonpeptide neurokinin (NK)1 antagonists display a marked difference in affinity for rat versus human NK1 receptors. The molecular basis for the species selectivity of RP67580 and CP96,345 has been previously addressed [J. Biol. Chem. 267:25668-25671 (1992); J. Biol. Chem. 268:2319-2323 (1993)]. We are extending these previous results to additional NK1 antagonists, which are members of different chemical families. Included is a new perhydroisoindolol, RPR100893, which unlike its parent compound (RP67580) is human receptor selective. Chimeric rat/human NK1 receptors, as well as rat and human mutant NK1 receptors, were constructed and expressed in COS-1 cells, and affinities for substance P and the various antagonists were determined in binding studies. With human receptor-selective antagonists, the rat R290(S-->I) mutation was the most effective in increasing antagonist affinity (from 7- to 23-fold). Combination with the R116(L-->V) mutation led to an additional increase in affinity for trans-4-hydroxy-1-(1H-indol-3-ylcarbonyl)-L-prolyl-N- methyl-N-(phenylmethyl)-L-tyrosineamide (a derivative of FK888) and to nearly full human receptor affinity for RPR100893 and (+/-)-CP99,994. Based on the gains in affinities, these results confirm and extend the role of residues 116 and 290 of the NK1 receptor in the species selectivity of these three new human receptor-selective NK1 antagonists. In comparison, the affinity of RP67580, the least selective molecule, was most affected by changes at position 116, and combination with mutations at either position 97 (V-->E) or position 290 led to the human receptor phenotype. For the heterosteroid KAN610857, modifications of the rat receptor at positions 97 and 290, and to a lesser degree position 116, were the most effective in reducing affinity. Two double-mutants [R(97,290) and R(116,290)], although different from those identified for RP67580, also displayed human receptor-like affinity. Therefore, the molecular determinants of the species selectivity appear to be different, in part, between rat and human receptor-selective compounds, even between closely related chemical families.